XAS STUDIES OF ACTIVE SITES AND COMPONENT INTERACTIONS IN BACTERIAL MULTICOMPONE

细菌复合体活性位点和成分相互作用的 XAS 研究

• 批准号: 7954221
• 负责人: KEITH O HODGSON
• 金额: $ 0.02万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954221
• 关键词: Active Sites; Affect; Bathing; Binding; Catalysis; Characteristics; Complex; Computer Retrieval of Information on Scientific Projects Database; Coupling; Dioxygen; Electronics; Enzymes; Family; Funding; Goals; Grant; Hydrocarbons; Institution; Iron; Methane hydroxylase; Mixed Function Oxygenases; Molecular; Oxidoreductase; Oxygen; Phenol 2-monooxygenase; Proteins; Pseudomonas stutzeri; Reaction; Research; Research Personnel; Resources; Role; Series; Site; Source; Spectrum Analysis; Structure; Substrate Specificity; System; United States National Institutes of Health; absorption; carboxylate; flexibility; genetic regulatory protein; novel; oxidation; protein protein interaction; research study; structural biology; synchrotron radiation; toluene 2-xylene monooxygenase

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We propose to probe systematically the electronic and geometric structure of the carboxylate-bridged nonheme diiron sites in a series of bacterial multicomponent monooxygenases (BMMs) using x-ray absorption spectroscopy (XAS). BMMs comprise a family of enzymes capable of utilizing molecular oxygen to oxidize a variety of hydrocarbon substrates, and require complexes between several protein components for efficient catalysis. Our goal is to elucidate the role of component interactions and structural flexibility at the diiron active center in the catalytic function and substrate specificity of BMMs. The proteins systems studied will include soluble methane monooxygenase (sMMO) from M. capsulatus (Bath), toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, and phenol hydroxylase (PH) from Pseudomonas stutzeri OX1. Our previous XAS studies of the sMMO and ToMO enzymes have afforded a structural characterization of the diiron site in the hydroxylase component, and identified its changes upon interaction with the regulatory proteins. We plan to extend XAS experiments to explore other protein-protein interactions in these systems and in the novel phenol hydroxylase. Specifically, we propose i) to obtain electronic and structural information on the diiron active center in intermediate species formed in the reaction of the hydroxylase component of BMMs with dioxygen; ii) to analyze the electronic and geometric structure of the dinuclear iron site in different oxidation states in the three hydroxylases; iii) to elucidate the effect of the coupling protein on the active center in the phenol hydroxylase; and iv) to determine how the binding between the hydroxylase and reductase component containing the [2Fe-2S] cluster affects the electronic and metrical characteristics of the dimetal active site and/or [2Fe-2S] center in the three multicomponent monooxygenases.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 我们建议使用X射线吸收光谱（XAS），在一系列细菌多组分单加氧酶（BMMS）中系统地探测羧酸桥桥接的非血红素二氢酶（BMMS）的电子和几何结构。 BMM包括一个能够利用分子氧氧化各种烃底物的酶家族，并需要几种蛋白质成分之间的复合物来有效催化。我们的目标是阐明在BMM的催化功能和底物特异性中，二龙活跃中心对组件相互作用和结构柔韧性的作用。所研究的蛋白质系统将包括来自M. capsulatus（BATH）的可溶性甲烷单氧酶（SMMO），来自Pseudomonas stutzeri ox1的甲苯/O-二甲苯单加仑酶（Tomo），以及来自pseudomonas stuteriaas stuterie ox1的苯酚羟化酶（pH）。我们先前对SMMO和TOMO酶的XAS研究对羟化酶成分中的二氮位点进行了结构表征，并确定了其与调节蛋白相互作用时的变化。我们计划扩展XAS实验，以探索这些系统和新型苯酚羟化酶中的其他蛋白质 - 蛋白质相互作用。具体而言，我们建议i）在BMMS羟化酶成分与二氧化物的反应中形成的中间物种中的二龙活性中心获取电子和结构信息； ii）分析三种羟化酶中不同氧化态中双核铁部位的电子和几何结构； iii）阐明偶联蛋白对苯酚羟化酶活性中心的影响；和iv）确定包含[2FE-2S]簇的羟化酶和还原酶分量之间的结合如何影响三个多核分单加速率的二胺活性位点和/或[2FE-2S]中心的电子和度量特性。